Method for inhibiting reperfusion injury using antibodies to P-selectin glycoprotein ligand

ABSTRACT

P-selectin has been demonstrated to bind primarily to a single major glycoprotein ligand on neutrophils and HL-60 cells, when assessed by blotting assays and by affinity chromatography of [ 3 H]glucosamine-labeled HL-60 cell extracts on immobilized P-selectin. This molecule was characterized and distinguished from other well-characterized neutrophil membrane proteins with similar apparent molecular mass. The purified ligand, or fragments thereof (including both the carbohydrate and protein components), or antibodies to the ligand, or fragments thereof, can be used as inhibitors of binding of P-selectin to cells.

This is a continuation of U.S. Ser. No. 09/635,297, filed Aug. 9, 2000,now U.S. Pat. No. 6,309,639 which is a divisional of U.S. Ser. No.09/207,375, filed Dec. 8, 1998, now U.S. Pat. No. 6,177,547, issued onJan. 23, 2001; which is a continuation of 08/438,280, filed May 10,1995, now U.S. Pat. No. 5,852,175, issued on Dec. 22, 1998; which is adivisional of U.S. Ser. No. 08/278,551, filed Jul. 21, 1994, now U.S.Pat. No. 5,464,778, issued on Nov. 7, 1995; which is a continuation ofU.S. Ser. No. 07/976,552, filed Nov. 16, 1992, now abandoned; which is acontinuation-in-part of U.S. Ser. No. 07/650,484, filed Feb. 5, 1991,now abandoned.

BACKGROUND OF THE INVENTION

The United States government has rights in this invention as a result ofNational Institutes of Health grants HI. 34363 (R. P. McEver) and HL45510 (R. P. McEver and K. L. Moore), CA. 38701 (A. Varki), IT4 RR 05351(R. D. Cummings), and GM 45914 (D. F. Smith).

P-selectin (CD62, GMP-140, PADGEM), a Ca²⁺-dependent lectin on activatedplatelets and endothelium, functions as a receptor for myeloid cells byinteracting with sialylated, fucosylated lactosaminoglycans. P-selectinbinds to a limited number of protease-sensitive sites on myeloid cells,but the protein(s) that carry the glycans recognized by P-selectin areunknown. Blotting of neutrophil or HL-60 cell membrane extracts with[¹²⁵I] P-selectin and affinity chromatography of [³H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectinligands. A major ligand was identified with an approximately 250,000M_(r) under nonreducing conditions and approximately 120,000 underreducing conditions. Binding of P-selectin to the ligand was Ca²⁺dependent and was blocked by mAbs to P-selectin. Brief sialidasedigestion of the ligand increased its apparent molecular weight;however, prolonged digestion abolished binding of P-selectin.Peptide:N-glycosidase F treatment reduced the apparent molecular weightof the ligand by approximately 3,000 but did not affect P-selectinbinding. Western blot and immunodepletion experiments indicated that theligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le^(x),nor was it leukosialin, a heavy sialylated glycoprotein of similarmolecular weight. The preferential interaction of the ligand withP-selectin suggests that it may play a role in adhesion of myeloid cellsto activated platelets and endothelial cells.

The selectins are three structurally related membrane glycoproteins thatparticipate in leukocyte adhesion to vascular endothelium and platelets,as reviewed by McEver, “Leukocyte interactions mediated by selecting”Thromb. Haemostas. 66:80-87 (1991). P-selectin (CD62), previously knownas GMP-140 or PAD-GEM protein, is a receptor for neutrophils, monocytesand subsets of lymphocytes that is rapidly translocated from secretorygranule membranes to the plasma membrane of activated platelets, asreported by Hamburger and McEver, “GMP 140 mediates adhesion ofstimulated platelets to neutrophils” Blood 75:550-554 (1990); Larsen etal., “PADGEM protein: a receptor that mediates the interaction ofactivated platelets with neutrophils and monocytes” Cell 59:305-312(1989) and endothelial cells, as reported by Geng et al., “Rapidneutrophil adhesion to activated endothelium mediated by GMP-140” Nature343:757-760 (1990); Lorant et al., “Coexpression of GMP 140 and PAF byendothelium stimulated by histamine or thrombin: A juxtacrine system foradhesion and activation of neutrophils” J. Cell Biol. 115:223-234(1991).

E-selectin (ELAM-1) is a cytokine-inducible endothelial cell receptorfor neutrophils, as reported by Bevilacqua et al., “Identification of aninducible endothelial leukocyte adhesion molecule” Proc. Natl. Acad.Sci. USA 84:9238-9242 (1987), monocytes, as reported by Hession et al.,“Endothelial leukocyte adhesion molecule 1: Direct expression cloningand functional interactions” Proc. Natl. Acad. Sci. USA 87:1673-1677(1990), and memory T cells, as reported by Picker et al., “ELAM-1 is anadhesion molecule for skin homing T cells” Nature (London) 349:796-799(1991); Shimizu et al., “Activation-independent binding of human memoryT cells to adhesion molecule ELAM 1” Nature (London) 349:799-802 (1991).L-selectin (LAM-1, LECAM-1), a protein expressed on myeloid cells andmost lymphocytes, participates in neutrophil extravasation intoinflammatory sites and homing of lymphocytes to peripheral lymph nodes,as reported by Lasky et al., “Cloning of a lymphocyte homing receptorreveals a lectin domain” Cell 56:1045-1055 (1989); Siegelman et al.,“Mouse lymph node homing receptor cDNA clone encodes a glycoproteinrevealing tandem interaction domains” Science (Wash. DC) 243:1165-1172(1989); Kishimoto et al., “Neutrophil Mac-1 and MEL-1 adhesion proteinsinversely regulated by chemotactic factors” Science (Wash. DC)245:1238-1241 (1989); Watson et al., “Neutrophil influx into aninflammatory site inhibited by a soluble homing receptor IgG chimaera”Nature (London) 349:164-167 (1991).

Each selectin functions as a CA²⁺-dependent lectin by recognition ofsialylated glycans. Both E- and P-selectin interact with sialylated,fucosylated lactosaminoglycans on opposing cells, including the sialylLe^(x) tetrasaccharide, as reported by Phillips et al., “ELAM 1 mediatescell adhesion by recognition of a carbohydrate ligand, sialy-Le^(x) ”Science (Wash DC) 250:1130-1132 (1990); Walz et al., “Recognition byELAM-1 of the sialyl-Le^(x) determinant on myeloid and tumor cells”Science (Wash. DC) 250:1132-1135 (1990); Lowe et al., “ELAM 1 dependentcell adhesion to vascular endothelium determined by a transfected humanfucosyltransferase cDNA” Cell 63:475-484 (1990); Tiemeyer et al.,“Carbohydrate ligands for endothelial-leukocyte adhesion molecule 1”Proc. Natl. Acad. Sci. USA 88:1138-1142 (1991); Goelz et al., “ELFT: agene that directs the expression of an ELAM-1 ligand” Cell 63:1349-1356(1990); Polley et al., “CD62 and endothelial cell-leukocyte adhesionmolecule 1 (ELAM 1) recognize the same carbohydrate ligand sialyl-Lewisx” Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991); Zhou et al., “Theselectin GMP-140 binds to sialyated, fucosylated lactosaminoglycans onboth myeloid and nonmyeloid cells” J. Cell Biol. 115:557-564 (1991).However, the precise carbohydrate structures on myeloid cells recognizedby these two selectins under physiologic conditions are not known. Suchligands might have unique structural features that enhance the bindingspecificity and/or affinity for their respective receptors.

P-selectin isolated from human platelets binds with apparent highaffinity to a limited number of sites on neutrophils (Moore et al., “GMP140 binds to a glycoprotein receptor on human neutrophils: evidence fora lectin-like interaction” J. Cell Biol. 112:491-499 (1991); Skinner etal., “GMP-140 binding to neutrophils is inhibited by sulfated glycans”.J. Biol. Chem. 266:5371-5374 (1991) and HL-60 cells (Zhou et al., “Theselectin GMP-140 binds to sialyated, fucosylated lactosaminoglycans onboth myeloid and nonmyeloid cells” J. Cell Biol. 115:557-564 (1991)).Binding is abolished by treatment of the cells with proteases (Moore etal., 1991), suggesting that the glycans on myeloid cells recognizedpreferentially by P-selectin are on glycoprotein(s) rather than onglycolipids. The number of binding sites for platelet P-selectin onneutrophils has been estimated at 10,000-20,000 per cell (Moore et al.,1991; Skinner et al., 1991), suggesting that these sites constitute asmall component of the total cell surface protein. The protein portionof this ligand(s) may be crucial for binding by presenting the glycan inan optimal configuration, clustering glycans to enhance avidity,favoring the formation of specific oligosaccharide structures bycellular glycosyltransferases or modifying enzymes, and/or stabilizingthe lectin-carbohydrate interaction through protein-protein interactionswith P-selectin.

The potential importance of protein components in enhancing ligandaffinity is supported by studies of. CHO cells transfected with aspecific fucosyltransferase (Zhou et al., 1991). These cells expresshigher amounts of the sialyl Le^(x) antigen than do HL-60 cells and haveprotease-sensitive binding sites for P-selectin. However, theinteraction of P-selectin with these sites is of much lower apparentaffinity than with those on myeloid cells, and adhesion of transfectedCHO cells to immobilized P-selectin is weaker than that of neutrophilsand HL-60 cells (Zhou et al., 1991). These observations suggest thatmyeloid cells express one or more membrane glycoproteins not found onCHO cells that enhance the lectin-mediated interaction with P-selectin.Alternatively, myeloid cells may express a glycosyltransferase ormodifying enzyme not present in CHO cells.

It is therefore an object of the present invention to identify andcharacterize a specific glycoprotein ligand for P-selectin (CD62).

It is a further object of the present invention to provide methods andcompositions derived from the characterization of a specificglycoprotein ligand for P-selectin for use in modifying inflammatoryprocesses and in diagnostic assays.

SUMMARY OF THE INVENTION

P-selectin has been demonstrated to bind primarily to a single majorglycoprotein ligand on neutrophils and HL-60 cells, when assessed byblotting assays and by affinity chromatography of [³H]glucosamine-labeled HL-60 cell extracts on immobilized P-selectin. Thismolecule was characterized and distinguished from otherwell-characterized neutrophil membrane proteins with similar apparentmolecular mass.

The purified ligand, or fragments thereof (including both thecarbohydrate and protein components), or antibodies to the ligand, orfragments thereof, can be used as inhibitors of binding of P-selectin tocells.

DETAILED DESCRIPTION OF THE INVENTION

U.S. Ser. No. 07/554,199 filed Jul. 17, 1990 entitled “PeptidesSelectively Interacting with Selectins” by Rodger P. McEver, describedthe ability of P-selectin (GMP-140) to mediate cell-cell contact bybinding to carbohydrate ligands on target cells and specific binding toprotease-sensitive sites on human neutrophils. Studies with antibodiesand with neuraminidase indicated that P-selectin bound to carbohydratestructures related to sialylated, fucosylated lactosaminoglycans. Asdescribed in U.S. Ser. No. 07/650,484 entitled “Ligand for GMP-140Selectin and Methods of Use Thereof” filed Feb. 5, 1991 by Rodger P.McEver, P-selectin was also demonstrated to bind to sialylated,fucosylated lactosaminoglycans (including the tetrasaccharide sialylLewis x (sLe^(x))) on both myeloid and nonmyeloid cells.

The ability of proteases to abolish P-selectin binding to neutrophilsindicated that high affinity binding of P-selectin to myeloid cellsoccurred through interactions with cell surface glycoprotein(s) ratherthan with glycolipids. As also described in U.S. Ser. No. 07/650,484,P-selectin bound preferentially to a glycoprotein in human neutrophilextracts of M_(r) 120,000, as analyzed by SDS-PAGE under reducingconditions. The glycoprotein was partially purified on a P-selectinaffinity column. It appeared to be heavily glycosylated because itstained poorly with silver and Coomassie blue. It appeared to be heavilysialylated because it bound to a wheat germ agglutinin affinity column.Treatment of the glycoprotein ligand with low doses of sialidase slowedits mobility on SDS gels, a pattern consistent with partialdesialylation of heavily O-glycosylated proteins. Binding of P-selectinto the glycoprotein ligand was Ca²⁺-dependent, blocked by monoclonalantibodies to P-selectin that also block P-selectin binding toleukocytes, and abolished by extensive treatment of the ligand withsialidase.

The preferential binding of P-selectin to the 120,000 D glycoproteinligand in myeloid cell extracts suggested that it contained specialstructural features that are recognized with high affinity byP-selectin. Such structures might not be present on every protein orlipid characterized by sialylated, fucosylated structures such assLe^(x). It has now been further demonstrated that the adhesion ofmyeloid cells to immobilized P-selectin is much stronger than that toNeoLewis CHO cells (a cell line expressing sialylated, fucosylatedlactosaminoglycans, described in U.S. Ser. No. 07/650,484), even thoughthe NeoLewis cells express higher levels of sLe^(x) antigen, as reportedby Zhou et al., J. Cell Biol. 115:557-564 (1991). Furthermore,fluid-phase [¹²⁵I ]P-selectin binds with high affinity to a limitednumber of sites on myeloid cells, whereas it binds with lower affinityto a higher number of sites on NeoLewis CHO cells. The 120,000 Dglycoprotein ligand for P-selectin in neutrophil extracts is likely tocorrespond to the limited number of protease-sensitive, high affinitybinding sites for P-selectin on intact neutrophils. Interaction ofP-selectin with these sites may be required for efficient adhesion ofleukocytes in flowing blood to P-selectin expressed by activatedplatelets or endothelial cells.

Further structural features of the glycoprotein ligand for P-selectinand a method for purifying the ligand are described below. The purifiedligand, or fragments thereof (including both the carbohydrate andprotein components), or antibodies to the ligand, or fragments thereof,can be used as inhibitors of binding of P-selectin to cells.

MATERIALS AND METHODS Materials

Wheat germ agglutinin (WGA)-agarose, pepstatin, aprotinin,N-acetylglucosamine, leupeptin, antipain, benzamidine, MOPS, Pipes, BSA,EDTA, EGTA, and Ponceau S were purchased from Sigma Chemical Co. (St.Louis, Mo.). Diisopropylfluorophosphate, dichloroisocoumarin, TritonX-100 (protein grade), and sialidase (neuraminidase) from Arthrobacterureafaciens (75 U.mg, EC 3.2.1.18) were obtained from Calbiochem-BehringCorp. (La Jolla, Calif.), Micro BCA protein assay kits and Lubrol PX(Surfact Amp PX) were purchased from Pierce Chemical Company (Rockford,Ill.). Enzymobeads™, Tween-20, Affigel™-15, and high molecular weightprotein standards were from Bio Rad Laboratories (Richmond, Calif.).Endo-β-galactosidase (150 U/mg, EC 3.2.1.103) from Bacteroides fragilis,4-methyl-umbelliferyl a-N-acetyineuraminic acid, and2,3-dehydro-2,3-dideoxy-N-acetylneuraminic acid (Neu2en5Ac) wereobtained from Boehringer Mannheim Biochemicals (Indianapolis, Ind.).Peptide:N glycosidase F (PNGaseF) from Flavobacterium meningosepticum(EC 3.2.2.18, N-glycanase) and endo-a-N-acetylgalactosaminidase formDiplococcus pneumoniae (EC 3.2.1.97, O-glycanase™) were purchased fromGenzyme (Cambridge, Mass.). HBSS was obtained from Gibco Laboratories(Grand Island, N.Y.). Vecta-Stain ABC kits were purchased from VectorLaboratories Inc. (Burlingame, Calif.). Phycoerythrin-streptavidin wasobtained from Becton Dickinson & Co. (San Jose, Calif.) andphycoerythrin-conjugated anti-mouse IgG₁ was from Caltag (South SanFrancisco, Calif.). Rabbit anti-mouse IgG was purchased from OrganonTeknika (Durham, N.C.) and protein A-Sepharose CL4B was from PharmaciaFine Chemicals (Piscataway, N.J.). [6-³H]glucosamine was obtained fromDupont/New England Nuclear (Boston, Mass.). All other chemicals were ofthe highest grade available.

Antibodies and Proteins

The anti-P-selectin murine mAbs S12 and G1, and goat anti-humanP-selectin IgG were prepared and characterized as described by McEverand Martin, “A monoclonal antibody to a membrane glycoprotein binds onlyto activated platelets” J. Biol. Chem. 259:9799-9804 (1984); Geng etal., (1990); Lorant et al., (1991). Rabbit polyclonal antisera andmurine mAbs to human lamp-1 (CD3), described by Carlsson et al.,“Isolation and characterization of human lysosomal membraneglycoproteins, h-lamp-1 and h-lamp-2. Major sialoglycoproteins carryingpolylactosaminoglycan” J. Biol. Chem. 263:18911-18919 (1988), and lamp-2(BB6), Carlsson and Fukuda, “Structure of human lysosomal membraneglycoprotein 1. Assignment of disulfide bonds and visualization of itsdomain arrangment” J. Biol. Chem. 264:20526-20531 (1989), and rabbitpolyclonal anti-human leukosialin antiserum, described by Carlsson andFukuda, “Isolation and characterization of leukosialin, a majorsialoglycoprotein on human leukocytes” J. Biol. Chem. 261:12779-12786(1986) were provided by Dr. Sven Carlsson (University of Umea, Umea,Sweden). Anti-human leukosialin (CD43) mAb (Leu-22) was purchased fromBecton Dickinson & Co. (San Jose, Calif.). The anti-L-selectin murinemAb antibodies DREG-56, DREG-55, and DREG-200, described by Kishimoto etal., “Identification of a human peripheral lymph node receptor: arapidly down-regulated adhesion receptor” Proc. Natl. Acad. Sci. USA87:2244-2248 (1990) were provided by Dr. Takashi Kei Kishimoto(Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Conn.). AllmAbs are of the IgG₁ subtype and were used in purified form. Leukosialinpurified from HL 60 cells (Carlsson and Fukuda, 1986) was provided byDr. Sven Carlsson (University of Umea). P-selectin was purified fromhuman platelets as described by Moore et al., (1991). The teachings ofthese references are specifically incorporated herein.

Preparation of Membranes

Erythrocyte membranes were isolated from leukocyte-depleted humanerythrocytes as described by Rollins and Sims, “Thecomplement-inhibitory activity of CD59 resides in its ability to blockincorporation of C9 into membrane C5b9” J. Immunol. 144:347-3483 (1990)and extracted with 0.1 M NaCl, 10 mM MOPS, pH 7.5, 1% Lubrol™ PX.Detergent-insoluble material was removed by centrifugation at 16,000 gfor 10 min.

Human neutrophils isolated by discontinuous leukopheresis from volunteerdonors were purchased from the Oklahoma Blood Institute (Oklahoma City,Okla.). Each product contained 1.5-3.3×10₁₀ leukocytes (approximately850% neutrophils). The neutrophil product was centrifuged at 200 g for20 min and the platelet-rich plasma removed. Erythrocytes were lysed byresuspending the pellets with 5 mM EDTA, pH 7.5, in H₂O for 20 s. Anequal volume of 1.18% NaCl, 5 mN EDTA, pH 7.5, was then added to restoreisotonicity. The cells were centrifuged at 500 g for 5 min andresuspended in ice-cold HBSS containing 5 mM EDTA and 10 mM. MOPS, pH7.5. Diisopropylfluorophosphate was then added to a final concentrationof 2 mM and the cell suspension incubated for 10 min on ice. The cellswere centrifuged at 500 g for 5 min at 4° C. and resuspended in ice-cold100 mM KCl, 3 mM NaCl, 1 mM Na₂ATP, 3.5 mM MgCl₂, 10 mM Pipes pH 7.3(relaxation buffer). To this suspension the following proteaseinhibitors were added at the indicated final concentrations: 2 mMdiisopropylfluorophosphate, 20 μM leupeptin, 30 μM antipain, and 1 mMbenzamidine. The cell suspension was pressurized with N₂ at 350 psi in acell disruption bomb (model 4635; Parr Instrument Company, Moline, Ill.)for 40 min at 4° C. with constant stirring as described by Borregaard etal., “Subcellular localization of the b cytochrome component of thehuman neutrophil microbiocidal oxidase: Translocation during activation”J. Cell Biol. 97:52-61 (1983). The cavitate was collected into EGTA (2mM final concentration) and nuclei and undisrupted cells were pelletedat 500 g for 10 min at 4° C. The cavitate was fractionated as describedby Eklund and Gabig, “Purification and characterization of a lipidthiobis ester from human neutrophil cytosol that reversibly deactivatesthe O²-generating NADPH oxidase” J. Biol. Chem. 265:8426-8430 (1990).Briefly, it was layered over 40% sucrose in relaxation buffer containing2 mM EGTA, 20 μM leupeptin, 30 μM antipain, and 1 mM benzamidine, andcentrifuged at 104,000 g (at r_(av)) for 45 min at 4° C. in a rotor(model SW28; Beckman Instruments, Inc., Palo Alto, Calif.). The toplayer (FX₁), the 40% sucrose layer (FX₂), and the granule pellet (FX₃)were collected and assayed for lactate dehydrogenase as a cytoplasmicmarker, alkaline phosphatase as a plasma membrane marker, andmyeloperoxidase as a marker for azurophilic granules as described byBorregaard et al., (1983); Geng et al., (1990).

Table I shows the distribution of marker enzymes in the variousfractions. FX₂, enriched for alkaline phosphatase, was diluted with fourvolumes of 0.1 M NaCl, 10 mM MOPS, pH 7.5, and centrifuged at 111,000 g(at r_(av)) for 60 min at 4° C. in a rotor (model 50.2 Ti; BeckmanInstruments, Inc.). The supernatant was collected and the membranepellet was extracted with 1% Lubrol™ PX, 0.1 M NaCl, 10 mM MOPS, pH 7.5,0.02% sodium azide, 20 μM leupeptin, 30 μM antipain, 1 mM benzamidine,and stored at 4° C.

HL-60 cells, maintained in suspension culture in RPMI-1640 supplementedwith 10% FCS, 100 IU/ml penicillin, and 100 μg/ml streptomycin, werewashed in HBSS, 10 mM MOPS, pH 7.5, and membranes were isolated exactlyas described for neutrophils.

Partial Purification of P-selectin Ligand

Neutrophil or HL-60 cell membrane extracts were applied to a wheat germagglutinin (WGA) affinity column (0.9×20 cm. 7.6 mg lectin/ml resin)equilibrated at room temperature with 0.5 M. NaCl, 10 mM MOPS, pH 7.5,0.02% sodium azide, 0.1% Lubrol™PX. The column was washed with fivecolumn volumes of equilibration buffer, followed by-two column volumesof 0.1 M NaCl, 10 mM MOPS, pH 7.5, 5 mM EDTA, 0.02% sodium azide, 0.01%Lubrol™ PX. The column was then eluted with the above buffer containing100 mM N-acetylglucosamine. Protein-containing fractions were pooled andextensively dialyzed against 0.1 M NaCl, 10 mM MOPS, pH 7.5, 0.0.2%sodium azide, 0.01% Lubrol™ PX at 4° C. The dialyzed WGA column eluatewas made 1 mM in CaCl₂ and MgCl₂ and applied to a human serum albuminAffigel™-15 precolumn (0.9×11 cm, 25 mg protein/ml resin) hooked inseries to a P-selectin-Affigel™-15 column (0.6×13 cm, 2 mg protein/mlresin). The columns were equilibrated with 0.1 M NaCl, 10 mM MOPS, pH7.5, 1 mM Cacl₂, 1 mM MgCl₂, 0.02% sodium azide, 0.01% Lubrol™ PX. Afterthe samples were applied the columns were washed with 100 column volumesof equilibration buffer, and eluted with equilibration buffer containing5 mM EDTA. Yields were estimated by protein assays with the Micro BCAprotein assay kit using BSA as a standard.

TABLE I Distribution of Marker Enzymes from Subcellular Fractions ofNitrogen-cavitated Human Neutrophils. Lactate Alkaline dehydrogenaseMyeloperoxidase phosphatase FX₁ (cytosol) 95.6 ± 0.5 0 29.0 ± 2.7 FX₂(membrane)  4.1 ± 0.5  2.6 ± 1.0 56.8 ± 8.7 FX₃ (granule) 0 97.4 ± 1.014.1 ± 5.5 Results are expressed as the percentage of the total enzymeactivity in the cavitate (mean ± SD, n = 3).

P-selectin Blotting Assay

Samples were electrophoresed on 7.5% SDS polyacrylamide gels andproteins electrophoretically tranferred to Immobilon-P™ membranes(Millipore Corp., Bedford, Mass.) for 4-5 h at 0.5 A. The positions ofthe molecular weight standards were marked with a pen after staining themembranes with Ponceau S. The membranes were blocked overnight at 4° C.in 0.1 M NaCl, 10 mM MOPS, pH 7.5, 1 mM CaCl₂, 1 mM MgCl₂, 0.02% sodiumazide, 10% (wt/vol) Carnation™ nonfat dry milk, and then washed with thesame buffer containing 0.11% Tween-20 without milk. The membranes wereincubated with [¹²⁵I]P-selectin (0.5-1.0 nM), iodinated as described byMoore et al., 1991 using standard techniques, in 0.1 M NaCl, 10 mM MOPS,pH 7.5, 1 mM CaCl₂, 1 mM MgCl₂, 0.05% Lubrol™ PX, 1% human serum albuminfor 1 h at room temperature. After extensive washing the membrane wasdried and exposed to Kodak X-OMAT AR film (Eastman Kodak Company,Rochester, N.Y.) for 6 18 at −70° C. All the [¹²⁵I]P-selectin blotsshown are autoradiograms of the entire blot, corresponding to the areafrom the stacking gel interface to beyond the dye front on the originalgel.

Metabolic Radiolabeling of HL-60 Cells and Isolation of[³H]glucosamine-labeled P-selectin Ligand

HL-60 cells (1-2×10⁶ cells/ml) in 100-mm tissue culture dishes werelabeled for 48 h with 50 μCi/ml [6-³H]glucosamine at 37° C. in RPMI-1640containing 10% FCS, 2 mM glutamine, 100 IU/mI penicillin, and 100 μg/mlstreptomycin. At the end of the labeling periods the cells were washedthree times by centrifugation and resuspension in ice-cold PBS. The cellpellet was solubilized with 0.1 M NaCl, 10 mM MOPS, pH 7.5, 4 mM CaCl₂,4 mM MgCl₂, 1% Triton X-100, 20 μg/ml aprotinin, 20 μg/ml leupeptin, 8μg/ml pepstatin, 2 mM PMSF, 10 mM benzamidine, and 0.5 mMdichloroisocoumarin. The solubilized cells were allowed to sit on icefor 1-2 h and then sonicated for 20 min at 4° C. in a water bathsonicator. The cell extract was centrifuged for 5 min at 16,000 g andthe supernatant was applied to a P-selectin-Affigel™-15 column (0.25×13cm, 2 mg protein/ml resin) equilibrated with 0.1 M NaCl, 10 mM MOPS, pH7.5, 2 mM CaCl₂, 2mM MgCl₂, 0.1% Triton X 100. The column was washedwith 10-20 column volumes of equilibration buffer and bound material waseluted with equilibration buffer containing 10 mM EDTA. Fractions (1 ml)were collected and monitored for radioactivity by liquid scintillationcounting.

Analysis of [³H]glucosamine-labeled P-selectin Ligand

Metabolically labeled proteins eluted from the P-selectin column wereprecipitated in the presence of 0.1 mg/ml BSA by addition of cold TCA(10% final concentration). The resulting pellets were washed with 1 mlacidified acetone (0.2%), solubilized in 0.1 M NaOH and electrophoresedunder reducing and nonreducing conditions on 10% SDS-polyacrylamidegels. The gels were stained with Coomassis blue and then processed forfluorography with EN³HANCE (Dupont/New England Nuclear, Boston, Mass.)according to the manufacturer's instructions. The dried gels were thenexposed to Kodak X-OMAT AR film at −80° C.

Enzyme Digestion

In certain experiments, samples analyzed by P-selectin blotting were.pretreated with exo- or endo-glycosidases before SDS-PAGE. For sialidaseand endo-β-galactosidase digestions of P-selectin ligand, samples weredialyzed against 0.15 M NaCl, 50 mM acetate, pH 6.0, 9 mM CaCl₂, 0.02%azide, 0.01% Lubrol™ PX, and incubated for various times at 37° C. inthe presence or absence of 200 mU/ml of enzyme. For PNGaseF andendo-a-N-acetygalactosaminidase digestions, samples were first reducedand denatured by boiling in 0.5% SDS, 0.5% β-mercaptoethanol for 5 min,and then a 7.5-fold molar excess of NP-40 was added. The samples wereincubated for 13 h at 37° C. with either PNGaseF (20 U/ml at pH 8.6) orendo-a-N acetylagalactosaminidase (70 mU/ml at pH 6.5) in the presenceof 5 mM PMSF and 5 mM 1,10 phenanthroline.

Affinity purified [³H]glucosamine-labeled P-selectin ligand wasincubated for 24 h in 25 mM sodium acetate, pH 5.5 at 37° C. under atoluene atmosphere in the presence or absence of 1 U/mi of A.ureafaciens sialidase for 18 h. For PNGaseF digestion of metabolicallylabeled ligand, samples were denatured by boiling in 0.25% SDS 25 mMβ-mercaptoethanol for 5 min, and NP-40 was added in eight-fold excess(wt/wt) over SDS. The samples were incubated for 24 h with PNGaseF (3.3U/mI) in a toluene atmosphere. The samples were then precipitated withTCA and subjected to SDS-PAGE and fluorography as described above.

Flow Cytometry

Human neutrophils, isolated as described by Hamburger and McEver,(1990), were suspended (10⁶/ml) in HBSS containing 1% FCS and 0.1%sodium azide (HBSS/FCS/Az). 1 ml of neutrophil suspension was underlaidwith 100 μl FCS and centrifuged at 500 g for 5 min. The neutrophilpellet was resuspended in 50 μl of purified P selectin (10 μl/ml, inHBSS/FCS/Az), and then incubated sequentially with 50 μl ofbiotin-conjugated S12 (10 μg/ml, in HBSS/FCS/Az) and 20 μl ofphycoerythrin-streptavidin (neat). In certain experiments, theneutrophils were preincubated for 10-15 min with antisera or antibodiesbefore the addition of P-selectin. Between each step the cells werediluted with one ml of HBSS/FCS/Az, underlaid with 100 μl FCS, andcentrifuged at 500 g for 5 min. All steps were performed at 4° C. Afterthe last wash, the cells were fixed with 1 ml of 1% paraformaldehyde inHBSS and analyzed in a FACScan flow cytometer (FACScan is a registeredtrademark of Becton Dickinson & Co., Mountain View, Calif.) formattedfor two color analysis as described by Moore, et al., (1991). Binding ofP-selectin to intact neutrophils as assessed by this assay wasCa²⁺-dependent, was blocked by G1, and was abolished by pretreatment ofthe cells with trypsin or sialidase.

Immunoprecipitations

WGA eluate was incubated with 10 μg of anti-leukosialin (Leu22) or anisotype matched control monoclonal antibody for 1 h at 37° C. Themixture was then incubated with protein A-Sepharose CL4B beads saturatedwith rabbit anti-mouse IgG for 1 h at 37° C. The beads were pelleted,washed four times with 1 ml of 0.1 M NaCl, 20 mM Tris, pH 7.5, 1% TritonX-100, and bound material eluted by boiling 5 min in 2% SDS, 60 mM Tris,pH 6.8, and 5% β-mercaptoethanol. Immunoprecipitates andimmunosupernatants were then analyzed by P-selectin blotting and byWestern blotting using Leu22 as a probe.

Assay of Sialidase Activity in Commercial Enzyme Preparations

The sialidase activity in O-glycanase (endo-a N-acetylgalactosaminidase)or A. ureafaciens sialidase was assayed by incubation of dilutions ofthe enzymes with 50 nmol 4-methyl-umbelliferyl-a-N-acetyineuraminic acidin 50 μl of sodium cacodylate, pH 6.5, 10 mM calcium acetate, forvarious time periods. Incubations were quenched by addition of 0.95 ml0.1 M sodium bicarbonate, pH 9.3, and assayed for released4-methylumbellierone by fluorescence (excitation=365 nM, emission=450nM).

RESULTS Identification of a P-selectin Ligand

To identify proteins from myeloid cells which bind P-selectin,neutrophil and HL-60 cell membrane extracts were electrophoresed on 7.5%SDS-polyacrylamide gels, transferred to Immobilon membranes, and probedwith [¹²⁵I]P-selectin. When samples were analyzed without reduction,P-selectin bound preferentially to a glycoprotein species with anapproximately 250,000 M_(r) from both neutrophil and HL-60 cellmembranes as determined by SDS-PAGE. Cell membrane extracts (80 μgprotein/lane) were electrophoresed on 7.5% SDS-polyacrylamide gels undernonreducing or reducing conditions, transferred to Immobilon membranes,and probed with [¹²⁵I]P-selectin. Under nonreducing conditionsP-selectin also bound to proteins at the stacking gel interface and to aminor species with an approximately 160,000 M_(r). When samples wereanalyzed after reduction, P-selectin preferentially bound to aglycoprotein with an approximately 120,000 M_(r). Minor bands wereobserved at approximately 250,000 and approximately 90,000 M_(r). Underboth reducing and nonreducing conditions P-selectin also bound to theblots at the dye front. P-selectin binding proteins were not detectedwhen an equivalent amount of erythrocyte membrane protein was analyzedin parallel. The total proteins in the neutrophil cavitate were alsosolubilized with SDS and analyzed for their ability to interact withP-selectin with the blotting assay. P-selectin bound only to proteinswith apparent molecular weights of 120,000 and 90,000 under reducingconditions. Although the sensitivity of this analysis was limited by theamount of protein that could be run on the gel, the results indicatethat we did not exclude major ligands that were either not enriched inthe membrane fraction (FX₂) or not effectively solubilized by nonionicdetergent.

To further assess the specificity of the blotting assay, neutrophilmembrane extracts electrophoresed under reducing conditions were probedwith [¹²⁵I]P-selectin in the presence or absence of EDTA oranti-P-selectin mAbs. Neutrophil membrane extracts (200 μg protein/lane)were electrophoresed on 7.5% SDS-polyacrylamide gels under reducingconditions, transferred to Immobilon membranes, and probed with[¹²⁵I]P-selectin alone, in the presence of 10 mM EDTA, or in thepresence of 20 μg/ml of the anti-P selectin mAbs G1 or S12.[¹²⁵I]P-selectin binding to the major 120-kD and the minor 250-kDspecies was Ca²⁺-dependent, a characteristic of all selectin-dependentcellular interactions. Binding to both species was also blocked by G1, amAb to P-selectin that inhibits adhesion of myeloid cells to P-selectin,but not by S12, a mAb to P-selectin that does not block adhesion.Binding of [¹²⁵I]P-selectin was also inhibited by a 100-fold excess ofunlabeled P-selectin. The binding of [¹²⁵I]P-selectin to the dye frontand to the 90,000 D protein was not blocked by EDTA or G1, suggestionthat these interactions were nonspecific or used a specificCa²⁺-independent recognition mechanism.

Purification of P-selectin Ligand from Neutrophils

Neutrophils were disrupted and the membrane fraction (FX₂) isolated byfractionation of the cavitate as described in Materials and Methods. Themembrane fraction constituted approximately 5-7% (n>10) of the proteinin the cavitate. This fractionation depleted both cytosolic proteins andazurophilic granules (Table I). Proteins binding P-selectin were notdetected in the cytosolic fraction (FX₁) with the blotting assay. Thefinal membrane pellet was solubilized with nonionic detergent andapplied to a WGA column which bound 4-5% of the protein in the membraneextract. P-selectin blotting assays of reduced proteins demonstratedthat both the major 120,000 D and the minor 250,000. D ligands boundquantitatively to: WGA. However, the 90,000 D band and the band at thedye front observed in the membrane extract were not bound by WGA. Afterextensive dialysis, the WGA eluate was applied to an Affigel-15™precolumn in series with a P-selectin affinity column. Approximately 2%of the protein in the WGA eluate bound to the P-selectin column andcould be eluted with EDTA. Both the 250,000 D and the 120,000 D ligandsbound quantitatively to the P-selectin column. Quantitative analysis ofthe protein recovered from the P-selectin eluate indicated that theligand(s) formed less than 0.01% of the total protein in the neutrophilcavitate. Elution of bound proteins from the P-selectin column with EDTAdemonstrated that the interaction of nondenatured neutrophil ligandswith P-selectin was also Ca²⁺-dependent. Neither species was eluted fromthe Affigel-15 precolumn with EDTA.

A silver-stained SDS-polyacrylamide gel of proteins from the variousstages in the partial purification procedure was run under reducingconditions. Samples from the indicated steps of the isolation procedurewere electrophoresed on 7.5% SDS-polyacrylamide gels under reducingconditions, transferred to Immobilon membranes, and probed with[¹²⁵I]P-selectin. The amounts of protein loaded onto the lanes were asfollows: membrane extract and WGA flow through, 200 μg; WGA eluate andP-selecting flow through, 50 μg; P-selectin eluate, 2 μg. The samesamples (10 μg protein/lane) were also analyzed by SDS-PAGE under thereducing conditions followed by silver staining. The majorsilver-stained band in the P-selectin eluate had an approximately150,000 M_(r) which is similar to that of P-selectin itself. Todetermine whether this protein represented P-selectin that had leachedoff the P-selectin column, the P-selectin eluate was analyzed bySDS-PAGE under both reducing and nonreducing conditions, followed bysilver staining, Western blotting with goat anti-P-selectin IgG, andP-selectin blotting. The major silver-stained protein in the P-selectineluate was indeed P-selectin. Purified P-selectin migrates with anapproximately 120,000 M_(r) under nonreducing conditions; a minorcomponent migrates with an approximately 250,000 M_(r). After reductionthe protein migrates more slowly with an approximately 150,000 M_(r).The two nonreduced bands and the one reduced band detected by silverstaining of the P-selectin eluate co-migrated with purified P-selectinand were recognized by anti-P-selectin IgG. The P-selectin ligandidentified in the blotting assay was not detected by silver staining andmigrated differently than P-selectin under both reducing and nonreducingconditions. When the P-selectin eluate was electrophoresed withoutreduction, P-selectin did not bind to proteins at the stacking gelinterface. Therefore, the P-selectin binding proteins at the stackinggel interface, observed in extracts of neutrophil membranes, wereprobably an artifact due to the relatively high amount of protein loadedon the gel.

Characterization of the P-selectin Ligand

The ligand(s) on intact target cells requires sialic acids to interactwith P-selectin. To determine whether the ligand detected by blotting ofneutrophil membranes containedsialic acids that were essential forrecognition by P-selectin, neutrophil membrane glycoproteins which boundto WGA were treated with sialidase (200 mU/ml) for varying times beforeSDS-PAGE under reducing conditions and then analyzed for their abilityto bind P-selectin. Neutrophil WGA eluate (50 μg) was eithersham-treated or digested with 200 mU/mI of sialidase or with 20 U/ml ofPNGaseF for 16 h, then electrophoresed on 7.5% SDS polyacrylamide gelsunder reducing conditions, transferred to Immobilon membranes, andprobed with [¹²⁵I]P-selectin.

Sialidase digestion for 30 min increased the apparent molecular weightof the major 120,000 D ligand, a shift characteristic of heavilysialylated glycoproteins. Longer sialidase digestion did not furtheralter the electrophoretic mobility of the ligand but did abolish itsability to bind [¹²⁵I]P-selectin. Sialidase treatment had a similareffect on the minor 250 kD ligand.

These results demonstrate that the ligand(s) contains sialic acidresidues that are critical for recognition by P-selectin, but suggestthat only a portion of the sialic acid residues are required forbinding.

To examine whether the ligand contained N-linked glycans, neutrophilmembrane glycoproteins which bound to WGA were digested with PNGaseF.This treatment did not affect [¹²⁵I]P-selectin binding but did decreasethe apparent molecular weight of the ligand, consistent with theenzymatic removal of one or two N-linked-glycan chains. Thisdemonstrates that the ligand contains at least one N-linkedoligosaccharide chain that is not required for P-selectin binding.Although one could not directly assess whether N-linked glycans werequantitatively removed from the ligand, conditions that normally cleavesuch glycans from most proteins were used.

Prolonged treatment of neutrophil membrane extracts withendo-a-N-acetylgalactosaminidase (O-glycanase) abolished binding of[¹²⁵I]P-selectin in the blotting assay, whereas sham digestion waswithout effect. This was a surprising result, since only nonsialylatedGalβ1-3GalNAc disaccharides O-linked to serine or threonine residues areknown substrates for the enzyme. Assays using a synthetic sialidasesubstrate confirmed the presence of a small amount of sialidase (0.01mU/mU O-glycanase) contaminating the O-glycanase. Although the level ofactivity was small, it was stable to prolong ed incubations under theconditions recommended by the manufacturer for use of the O-glycanasepreparation. To prove that the contaminating sialidase was responsiblefor the loss of P-selectin binding, the digestions were repeated in thepresence of a competitive sialidase inhibitor, Neu2en5Ac. Under theseconditions endo-a-N-acetylgalacto saminidase digestion had no effect on[¹²⁵I]P-selectin binding to the ligand or the apparent molecular weightof the ligaen. Because the ligand requires sialic acid to interact withP-selectin, the blotting assay could not be used to assess the role ofO-linked glycans in recognition by P-selectin.

Isolation a of a P-selectin Ligand from Metabolically Labeled HL-60Cells

P-selectin blotting of denatu red membrane proteins from myeloid cellsmay not de tect molecules whose ability to bind P-selectin is dependenton secondary and/or tertiary structure. As an independent approach toidentify ligands for P-selectin, HL-60 cells were metabolically labeledwith [³ H]glucosamine, solubilized with nonionic detergent, and appliedto a P-selectin affinity column. After extensive washing, bound materialwas eluted with EDTA and analyzed by SDS-PAGE followed by fluorography.Samples were electrophoresed on 10% SDS polyacrylamide gels under bothnon reducing and reducing conditions and analyzed by fluorography. Othersamples were either sham treated or digested with 1 U/ml of sialidasefor 24 h or with 3.3. U/ml of PNGaseF for 24 h, and then electrophoresedon 10% SDS polyacrylamide gels under reducing conditions and analyzed byfluorography.

A single metabolically labeled species was eluted, which co-migratedunder both nonreducing and reducing conditions with the major speciesdetected in neutrophil and HL-60 cell membranes by blotting with[¹²⁵I]P-selectin. Only 0.15-0.5% of the total [³H]glucosamine-labeledHL-60 glycoproteins bound to the P-selectin column, indicating that theligand is not abundant. Sialidase treatment of the[³H]glucosamine-labeled P-selectin ligand from HL-60 cells produced thesame increase in apparent molecular weight that was observed for themajor neutrophil ligand identified by the P-selectin blotting assay. Inaddition, PNGaseF treatment caused the same decrease in the apparentmolecular weight of the HL-60 cell ligand that was observed for theneutrophil ligand.

Comparison of the P-selectin Ligand with Known Neutrophil MembraneProteins

The properties of the major 120,000 D P-selectin ligand were comparedwith those of three well-characterized neutrophil membrane proteins withsimilar apparent molecular weight. The first two molecules, lamp-1 andlamp-2, are abundant neutrophil proteins that are predominantlylocalized in lysosomal membranes but are also expressed in small amountson the cell surface. These proteins have a large number of complexN-linked glycan chains, many of which carry the sialyl Le^(x)tetrasaccharide. Polyclonal antisera (1:5 dilution) and mAbs (40 μg/ml)to lamp-1 (CD3) and lamp-2 (BB6) had no effect on binding of P-selectinto neutrophils as assessed by flow cytometry.

Membrane extracts (200 μg protein/lane) were electrophoresed on 7.5%SDS-polyacrylamide gels under nonreducing or reducing conditions,transferred to Immobilon membranes, and probed with [¹²⁵I]P-selectin ormurine monoclonal antibodies directed against human lamp-1 (CR3), humanlamp-2 (BB6), human L-selectin (DREG-200), or human leukosialin (Leu22).Western blot analysis of neutrophil membranes with mAbs to lamp-1 andlamp-2 showed that the electrophoretic mobilities of these proteinsunder nonreducing conditions were distinct from that of the P-selectinligand. In contrast to the P-selectin ligand, the electrophoreticmobilities of lamp-1 and lamp-2 are not affected by sialidase treatment.Although lamp-1 and lamp-2 from myeloid cells are rich inlactosaminoglycans sensitive to endo-β-galactosidase, treatment ofintact neutrophils with the enzyme did not affect binding of[¹²⁵I]P-selectin. Pretreatment of crude neutrophil membrane extracts orWGA column eluate with endo β-galactosidase (200 mU/ml, 1-2 h, 37° C.)also did not affect the apparent molecular weight of the ligand or itsability to bind [¹²⁵I]P-selectin. These data argue that lamp-1 andlamp-2 are not ligands for P-selectin even though they carry many sialylLe^(x) structures.

The third molecule whose apparent molecular weight is similar to the120,000 D P-selectin ligand is CD43 (leukosialin, sialophorin), aheavily sialylated membrane protein present on platelets and allleukocytes. It carries numerous O-linked sugar chains and isdifferentially glycosylated by cells of various hematopoietic lineages.Like the P-selectin ligand, treatment of leukosialin with sialidaseincreases its apparent molecular weight. However, in contrast to theP-selectin ligand, the electrophoretic mobility of leukosialin wasunaffected by reduction. Monospecific polyclonal anti-human leukosialinantisera (1:5 dilution) did not inhibit P-selectin binding toneutrophils as assessed by flow cytometry. Furthermore, immunodepletionof leukosialin from neutrophil membrane extracts did not depleteP-selectin ligand as assessed by the blotting assay. Finally,leukosialin purified from HL-60 cells did not bind P-selectin.Neutrophil WGA eluate (50 μg) and leukosialin purified from HL-60 cells(0.5 μg) were electrophoresed under reducing conditions on 7.5%SDS-polyacrylamide gels, transferred to Immobilon, and probed with[¹²⁵I]P-selectin. The same membrane was then probed with the monocionalanti-human leukosialin antibody Leu22.

Based on studies in which an antibody to L-selectin (DREG-56) partiallyinhibited neutrophil adhesion to P-selectin-transfected cells, it wassuggested that L-selectin is an important glycoprotein ligand on myeloidcells for P-selectin by Picker et al., “The neutrophil selectin LECAM-1presents carbohydrate ligands to the vascular selectins ELAM-1 andGMP-140” Cell 66:921-933 (1991). Although L-selectin is present inmembrane extracts and WGA eluates of neutrophil membranes, as detectedby Western blotting, [¹²⁵I]P-selectin did not bind to L-selectin in theblotting assay. In addition, the anti-L-selectin mAb DREG-56 (100 μg/ml)had no effect on the binding of purified P-selectin to quiescentneutrophils as assessed by flow cytometry. Neutrophils were preincubatedfor 15 min with buffer alone, 100 μ/ml of the anti-L-selectin monoclonalantibody DREG-56, or 100 μg/ml of the anti-P-selectin mAb GI beforeaddition of buffer or P-selectin. P-selectin binding was then detectedby sequential incubation of the cells with biotinylated S12 (anoninhibitory monoclonal antibody to P-selectin) andphycoerythrin-streptavidin as described in Materials and Methods.

Parallel control assays showed that the neutrophils expressed highlevels of L-selectin detactable by DREG-56. Binding of theanti-L-selectin mAb DREG-56 to the neutrophils was assessed by indirectimmunofluorescence using a phycoerythrin-conjugated anti-murine IgG₁antibody. Identical results were obtained with the anti-L-selectin mAbsDREG-55 and DREG-200. Thus, interactions with L-selectin do not appearto contribute to the binding of fluid-phase P-selectin to intactneutrophils or to immobilized proteins from neutrophil membraneextracts.

The following additional observations have been made relating to theligand.

Treatment of the ³H-glucosamine-labeled P-selectin ligand from HL-60cells with neuraminidase releases approximately 30% of the radioactivityas sialic acid.

Strong acid hydrolysis of the ³H-glucosamine-labeled P-selectin ligandfrom HL-60 cells releases both ³H-glucosamine and ³H-galactosamine inthe approximate ratio of 2:1, indicating that the ligand contains bothN-acetylglucosamine and N-acetylgalactosamine. In most glycoproteinsstructurally defined to date, the glucosamine and galactosamine occur asthe acetylated derivatives.

The presence of N-acetylgalactosamine is indicative of the presence ofO-linked oligosaccharides (or Ser/Thr-linked oligosaccharides) in whichGaINAc is commonly found in O-glycosidic a-linkage directly to aminoacid. The indicated presence of O-linked oligosaccharide is confirmed bythe further observation that the ligand binds quantitatively to theJacalin-Sepharose, an immobilized plant lectin that binds to the coredisaccharide sequence Galβ1-3GalNAco-Ser/Thr in glycoproteins.Jacalin-Sepharose can bind to O-linked oligosaccharides that havemodifications of this simple core. Thus, these results are not inconflict with the lack of sensitivity of the ligand to O-glycanase. Whenthe ³H-glucosamine-labeled P-selectin ligand from HL-60 cells is treatedwith mild base in the presence of sodium borohydride (50 mM NaOH, 1 MNaBH₄, 16 hr at 45° C.) to cause a beta-elimination reaction,approximately two-thirds of the radioactivity is released as amoderately sized oligosaccharide with more than four sugar residues, asdefined by chromatography on a column of BioGel P-10 in bicarbonatebuffer. It is well known from previous published studies thatoligosaccharides in O-glycosidic linkage, but not in N-glycosidiclinkage, are susceptible to release from peptide by this treatment.These results further support the observation that the ligand contains aconsiderable amount of O-linked oligosaccharides.

Treatment of the ³H-glucosamine-labeled P-selectin ligand from HL-60cells with O-glycanase and neuraminidase does not demonstrably affectthe apparent mobility of the ligand any more than neuraminidase alone.This indicates that the ligand does not contain large amounts of thesimple oligosaccharides in O-linkage to Ser/Thr residues. O-glycanase isan endoglycosidase which cleaves the sequence Galβ1-3GalNAca-Ser/Thr inglycoproteins to release the disaccharide Galβ1-3GalNAc. Treatment ofthe ³H-glucosamine-labeled P-selectin ligand from HL-60 cells withneuraminidase causes a decrease in the electrophoretic mobility of theligand.

The presence of numerous O-linked oligosaccharides on the ligand isconfirmed by the sensitivity of the ³H-glucosamine-labeled P-selectinligand from HL-60 cells to an enzyme termed O-glycoprotease. This enzymeis a protease which recognizes and cleaves peptide bonds withinglycoproteins that contain numerous sialic acid-containing O-linkedoligosaccharides.

The ligand contains the sialyl Lewis x (SLe^(x)) antigen(NeuAca2-3Galβ1-4 [Fuca1-3]GlcNAcβ-R). When the ³H-glucosamine-labeledP-selectin ligand from HL-60 cells was reapplied to a column ofP-selectin-Affigel™-15 it rebound. When this chromatography was done inthe presence of antibody to the SLe^(x) antigen (CSLEX1 monoclonalantibody (Fukushima, et al., Cancer Res. 44:5279-5285, 1984)), purchasedfrom Dr. Paul Teraski, University of California at Los Angeles, bindingwas more than 90% reduced. In contrast, when a control experiment wasdone in which the rechromatography occurred in the presence of antibodyto the Le^(x) antigen (which lacks sialic acid), there was little if anyeffect. The CSLEX1 anti-SLe^(x) antibody bound to the ligand as assessedby Western blotting.

The ligand contains poly-N-acetyllactosamine sequences of the type[3Galβ1-4GlcNAc β1]_(n). The ³H-glucosamine-labeled P-selectin ligandfrom HL-60 cells quantitatively binds to a column of immobilized tomatolectin, a plant lectin which has been shown to bind topoly-N-acetyllactosamine sequences within glycoproteins.

The ligand from HL-60 cells contains fucose. When HL-60 cells areincubated with 6-³H-fucose, the P-selectin ligand is radioactivelylabeled. All incorporated radioactivity is in fucose. Furthermore, whenthe ³H-fucose-labeled ligand is treated with mild base and sodiumborohydride to effect beta-elimination, ³H-fucose-labeledoligosaccharides are released that are both high molecular weight andmoderate molecular weight, as estimated by chromatography on a column ofBioGel P-10.

The differential mobility of the major ligand during SDS-PAGE in thepresence and absence of reducing agents suggests that the native ligandis a disulfide-linked homodimer or that a 120,000 D subunit is disulfidelinked to a distinct subunit that is not directly involved in P-selectinbinding. Since only a 120,000 D band was detected after electrophoresisof reduced P-selectin eluate from metabolically labeled HL-60 cells, aheterodimer would have to consist of nonidentical subunits with the sameapparent molecular weight and which undergo the same change inelectrophoretic mobility after sialidase and PNGaseF digestion.Alternatively, the 120-kD-labeled subunit would have to bedisulfide-linked to a subunit of similar apparent molecular weight thatis not labeled with [³H]glucosamine. A homodimeric ligand with twoequivalent binding sites might enhance the avidity of the interactionwith P-selectin. The ability of [¹²⁵I]P-selectin to bind to the ligandafter reduction and denaturation with SDS suggests that higher orderstructural features of the protein are not critical for recognition.

The blotting assay also detected two minorligands. The first has anapproximately 250,000 M_(r) under reducing conditions. Because itsmobility is identical to that of the major ligand under nonreducingconditions, it may represent a subpopulation of the major ligand that isresistant to reduction. The second has an approximately 160,000 M_(r)under nonreducing conditions. Binding of P-selectin to both minorligands was Ca²⁺ dependent and blocked by the mAb GI.

The isolation of a single glycoprotein from metabolically labeled HL-60cells suggests that P-selectin has a marked preference for a particularligand structure. L-selectin, which is expressed on leukocytes and bindsto sialylated structures on endothelial cells, interacts preferentiallywith 50,000 D and 90,000 D sulfated, fucosylated glycoproteins frommurine peripheral lymph nodes (Imai, et al., J. Cell Biol.113:1213-1222, 1991). Thus, both P-selectin and L-selectin appear tointeract with a small subset of glycoprotein ligands.

It has been demonstrated that L-selectin on neutrophils carries thesialyl Le^(x) epitope and that a mAb to L-selectin partially blocksneutrophil adhesion to cells transfected with P-selectin cDNA (Picker,it al., Cell 66:921-933, 1991). Based on these observations, it wasproposed that L-selectin on neutrophils is a predominant ligand forP-selectin. However, no direct interaction of L-selectin with P-selectinwas demonstrated. In the present study, binding of P-selectin toL-selectin in neutrophil membrane extracts was not detectable.Furthermore, the binding of P-selectin to intact neutrophils isunaltered by antibodies to L-selectin or by neutrophil activation thatcauses shedding of L-selectin from the cell surface. Although it isconceivable that L-selectin has weak affinity for P-selectin, thesignificance of this potential interaction remains to be established.

A recombinant P-selectin IgG chimera was shown to bind to myeloid cellsand to a sulfatide, Gal(3-SO₄) β1-Ceramide by Aruffo et al.,“CD62/P-selectin recognition of myeloid and tumor cell sulfatides” Cell67:35-44 (1991). Sulfatide also inhibited interaction of the chimerawith monocytoid U937 cells, as reported by Aruffo et al., (1991). It wasnot demonstrated whether binding of the P-selectin chimera to the cellsor to sulfatide was Ca²⁺ dependent, a fundamental characteristic ofselectin-dependent cellular interactions. Protease digestion of intactcells should increase the accessibility of P-selectin to potentialglycolipid ligands such as sulfatides. However, protease treatmentabolishes binding of P-selectin to neutrophils and HL-60 cells as wellas adhesion of neutrophils to immobilized P-selectin. In addition,although erythrocytes and platelets express sulfatides, they do notspecifically interact with P-selectin. Thus, it seems unlikely thatsulfatides are the principal mediators of adhesion of myeloid cells toP-selectin. It remains to be determined whether sulfatides inhibitbinding of P-selectin to myeloid cells by specific competition with aglycoprotein ligand or by indirect effects. It is possible that theP-selectin ligand described herein is sulfated or contains otherstructural features that are mimicked by sulfatides.

Previous studies by Zhou et al., (1991); and Polley et al., (1991) haveshown that P-selectin interacts with a(2-3) sialylated, a(1-3)fucosylated lactosaminoglycans, of which one is the sialyl Le^(x)tetrasaccharide. However, several observations suggest that the sialylLe^(x) tetrasaccharide per se does not bind with high affinity toP-selectin. First, some investigators (Moore et al., 1991; Aruffo etal., 1991; Polley, et al.), but not all, have found that sialyl Le^(x)inhibits interactions of myeloid cells with P-selectin. Second, CHOcells transfected with a fucosyltransferase express sialyl Le^(x) yetbind P-selectin with significantly lower affinity than do myeloid cells(Zhou et al., 1991). Third, HT-29 cells, which also express sialylLe^(x), do not interact at all with P-selectin (Zhou et al., 1991).Finally, several neutrophil membrane proteins known to carry the sialylLe^(x) structure, are distinct from the major glycoprotein ligandidentified herein and do not bind P-selectin in the assays describedhere. These observations suggest that the ligand contains structuralfeatures in addition to the sialyl Le^(x) tetrasaccharide that enhancethe affinity and/or specificity of its interaction with P-selectin.

A blotting assay of neutrophil and HL-60 cell membrane extracts was usedto search for ligands for P-selectin. As described previously in U.S.Ser. No. 07/650,484, [¹²⁵I]P-selectin bound preferentially to aglycoprotein of M_(r) 120,000 as assessed by SDS-PAGE under reducingconditions. Under nonreducing conditions, the ligand for P-selectin hadan apparent M_(r) of 250,000, suggesting that it is a disulfide-linkedhomodimer. In initial studies, the ligand was partially purified byserial affinity chromatography on wheat germ agglutinin (WGA) andP-selectin affinity columns. Proteins bound to the P-selectin columnwere eluted with EDTA. The glycoprotein ligand was greatly enriched inthe EDTA eluate from the P-selectin column, as assessed by the intensityof the band identified by [¹²⁵I]P-selectin blotting. As noted in U.S.Ser. No. 07/650,484, however, the ligand stained poorly with silver,consistent with its being an unusually heavily glycosylated protein. Inthe initial purifications, the only contaminating protein present notedby silver staining of the gel was a small amount of P-selectin itselfwhich had been leached from the affinity column. Using a new P-selectinaffinity column and more extensive washing procedures documented in themethods, the ligand has now been isolated free from contaminants. Thisconclusion is based on observation that there are no silver stainingbands present but the ligand is clearly identified by its ability tointeract with [¹²⁵I]P-selectin in the blotting assay.

As described in U.S. Ser. No. 07/650,484, partial removal of sialicacids with sialidase slowed the mobility of the ligand, a featurecharacteristic of heavily sialylated glycoproteins. Extensive sialidasedigestion abolished recognition of the ligand by P-selectin. It has nowbeen demonstrated that the ligand contain both N- and O-linkedoligosaccharides.

A form of the ligand in which the carbohydrate components areradiolabeled has also been purified by P-selectin affinitychromatography, as described above. SDS-PAGE analysis of the P-selectincolumn eluate, followed by fluorography, indicates that the only labeledprotein has an Mr of 250,000 under nonreducing conditions and 120,000under reducing conditions. The radiolabeled. ligand has the same shiftsin electrophoretic mobility following treatments with sialidase orPNGase F. Thus, all the features of the radiolabeled ligand correspondto those of the ligand identified by the P-selectin blotting assay.Because only a single radiolabeled species is isolated from theP-selectin affinity column, the carbohydrate structures of the ligandcan be analyzed in detail by procedures that have been developed, forexample, as reported by R. D. Cummings and S. Kornfeld, J. Biol. Chem257:11235-11240 (1982) and R. D. Cummings, et al., J. Biol. Chem. 258:15261-15273 (1983).

In summary, the glycoprotein ligand for P-selectin from myeloid cellshas the characteristics of a disulfide-linked homodimer with eachsubunit having an apparent Mr of 120,000 as assessed by SDS-PAGE. Theprotein has some N-linked carbohydrate but its most striking feature isthe presence of a large number of clustered O-linked glycans, most ofwhich appear to be larger than the usual simple O-linked chains cleavedby O-glycanase. Although the ligand contains the sLe^(x) structure, thedata indicate that additional structural features in the ligand arerequired to confer high affinity binding to P-selectin. These featuresmay include, but are not limited to, carbohydrate structures of morecomplexity than sLe^(x) itself, clustering of many glycan chains toincrease avidity, and specific orientations of the glycans relative tothe protein backbone.

Preparation of Diagnostic and Therapeutic Agents Derived from theProtein or Carbohydrate Components of the Glycoprotein Ligand forP-selectin

The glycoprotein ligand for P-selectin described above has a variety ofapplications as diagnostic reagents and, potentially, in the treatmentof numerous inflammatory and thrombotic disorders.

Diagnotic Reagents

Antibodies to the ligand can be used for the detection of humandisorders in which P-selectin ligands might be defective. Such disorderswould most likely be seen in patients with increased susceptibility toinfections in which leukocytes might not be able to bind to activatedplatelets or endothelium. Cells to be tested, usually leukocytes, arecollected by standard medically approved techniques and screened.Detection systems include ELISA procedures, binding of radiolabeledantibody to immobilized activated cells, flow cytometry,immunoperoxidase or immunogold analysis, or other methods known to thoseskilled in the arts.

Antibodies directed specifically to protein or carbohydrate componentsof the ligand can be use to distinquish defects in expression of thecore protein or in glycosyltransferases and/or modifying enzymes thatconstruct the proper oligosaccharide chains on the protein. Theantibodies can also be used to screen cells and tissues other thanleukocytes for expression of the protein or carbohydrate components ofthe ligand for P-selectin. Complementary DNA clones encoding the proteincomponent of the ligand can be isolated and sequenced. These probes canbe used as diagnostic reagents to examine expression of RNA transcriptsfor the ligand in leukocytes and other tissues by standard proceduressuch as Northern blotting of RNA isolated from cells and in situhybridization of tissue sections.

A similar approach can be used to determine qualitative or quantitativedisorders of P-selectin itself. The glycoprotein ligand, carbohydrates,or appropriate derivatives thereof, is labeled and tested for itsability to bind to P-selectin on activated platelets from patients withdisorders in which P-selectin might be defective.

The ligand, or components thereof, and also be used in assays ofP-selectin binding to screen for compounds that block interactions ofP-selectin with the ligand.

Clinical Applications.

Since P-selectin has several functions related to leukocyte adherence,inflammation, tumor metastases, and coagulation, clinically, compoundswhich interfere with binding of P-selectin and/or the other selecting,including E-selectin and L-selectin, such as the carbohydrates, can beused to modulate these responses. These compounds include the P-selectinligand, antibodies to the ligand, and fragments thereof. For example,the glycoprotein ligand, or components thereof, particularly thecarbohydrate moieties, can be use to inhibit leukocyte adhesion bycompetitively binding to P-selectin expressed on the surface ofactivated platelets or endothelial cells. Similarly, antibodies to theligand can be used to block cell adhesion mediated by P-selectin bycompetively binding to the P-selectin ligand on leukocytes or othercells. These therapies are useful in acute situations where effective,but transient, inhibition of leukocyte-mediated inflammation isdesirable. In addition, treatment of chronic disorders may be attainedby sustained administration of agents, for example, by subcutaneous ororal administration.

An inflammatory response may cause damage to the host if unchecked,because leukocytes release many toxic molecules that can damage normaltissues. These molecules include proteolytic enzymes and free radicals.Examples of pathological situations in which leukocytes can cause tissuedamage include injury from ischemia and reperfusion, bacterial sepsisand disseminated intravascular coagulation, adult respiratory distresssyndrome, tumor metastasis, rheumatoid arthritis and atherosclerosis.

Reperfusion injury is a major problem in clinical cardiology.Therapeutic agents that reduce leukocyte adherence in ischemicmyocardium can significantly enhance the therapeutic efficacy ofthrombolytic agents. Thrombolytic therapy with agents such as tissueplasminogen activator or streptokinase can relieve coronary arteryobstruction in many patients with severe myocardial ischemia prior toirreversible myocardial cell death. However, many such patients stillsuffer myocardial neurosis despite restoration of blood flow. This“reperfusion injury” is known to be associated with adherence ofleukocytes to vascular endothelium in the ischemic zone, presumably inpart because of activation of platelets and endothelium by thrombin andcytokines that makes them adhesive for leukocytes (Romson et al.,Circulation 67: 1016-1023, 1983). These adherent leukocytes can migratethrough the endothelium and destroy ischemic myocardium just as it isbeing rescued by restoration of blood flow.

There are a number of other common clinical disorders in which ischemiaand reperfusion results in organ injury mediated by adherence ofleukocytes to vascular surfaces, including strokes; mesenteric andperipheral vascular disease; organ transplantation; and circulatoryshock (in this case many organs might be damaged following restorationof blood flow).

Bacterial sepsis and disseminated intravascular coagulation often existconcurrently in critically ill patients. They are associated withgeneration of thrombin, cytokines, and other inflammatory mediators,activation of platelets and endothelium, and adherence of leukocytes andaggregation of platelets throughout the vascular system.Leukocyte-dependent organ damage is an important feature of theseconditions.

Adult respiratory distress syndrome is a devastating pulmonary disorderoccurring in patients with sepsis or following trauma, which isassociated with widespread adherence and aggregation of leukocytes inthe pulmonary circulation. This leads to extravasation of large amountsof plasma into the lungs and destruction of lung tissue, both mediatedin large part by leukocyte products.

Two related pulmonary disorders that are often fatal are inimmunosuppressed patients undergoing allogeneic bone marrowtransplantation and in cancer patients suffering from complications thatarise from generalized vascular leakage resulting from treatment withinterleukin-2 treated LAK cells (lymphokine-activated lymphocytes). LAKcells are known to adhere to vascular walls and release products thatare presumably toxic to endothelium. Although the mechanism by which LAKcells adhere to endothelium is not known, such cells could potentiallyrelease molecules that activate endothelium and then bind to endotheliumby mechanisms similar to those operative in neutrophils.

Tumor cells from many malignancies (including carcinomas, lymphomas, andsarcomas) can metastasize to distant sites through the vasculature. Themechanisms for adhesion of tumor cells to endothelium and theirsubsequent migration are not well understood, but may be similar tothose of leukocytes in at least some cases. Specifically, certaincarcinoma cells have been demonstrated to bind to both E-selectin, asreported by Rice and Bevilacqua. Science 246:1303-1306 (1991), andP-selectin, as reported by Aruffo, et al., Proc. Natl. Acad. Sci. USA89:2292-2296 (1992). The association of platelets with metastasizingtumor cells has been well described, suggestion a role for platelets inthe spread of some cancers. Since P-selectin is expressed on activatedplatelets, it is believed to be involved in association of plateletswith at least some malignant tumors.

Platelet-leukocyte interactions are believed to be important inatherosclerosis. Platelets might have a role in recruitment of monocytesinto atherosclerotic plaques; the accumulation of monocytes is known tobe one of the earliest detectable events during atherogenesis. Ruptureof a fully developed plaque may not only lead to platelet deposition andactivation and the promotion of thrombus formation, but also the earlyrecruitment of neutrophils to an area of ischemia.

Another area of potential application is in the treatment of rheumatoidarthritis.

In these clinical applications, the glycoprotein ligand, or fragmentsthereof, can be administered to block selectin-dependent interactions bybinding competitively to P-selectin expressed on activated cells. Inparticular, carbohydrate components of the ligand, which play a key rolein recognition by P-selectin, can be administered. Similarly, natural orsynthetic analogs of the ligand or its fragments which bind toP-selectin can also be administered. In addition, antibodies to theprotein and/or carbohydrate components of the ligand, or fragmentsthereof, can be administered. The antibodies are preferably of humanorigin or modified to delete those portions most likely to cause animmunogenic reaction. Carbohydrate components of the ligand or theantibodies, in an appropriate pharmaceutical carrier,; are preferablyadministered intravenously where immediate relief is required. Thecarbohydrate(s) can also be administered intramuscularly,intraperitoneally, subcutaneously, orally, as the carbohydrate,conjugated to a carrier molecule, or in a drug delivery device. Thecarbohydrate can be modified chemically to increase its in vivohalf-life.

The carbohydrate can be isolated from cells expressing the carbohydrate,either naturally or as a result of genetic engineering as described inthe transfected COS cell examples, or, preferably, by synthetic means.These methods are known to those skilled in the art. In addition, alarge number of glycosyltransferases have been cloned (J. C. Paulson andK. J. Colley, J. Biol. Chem. 264:17615-17618, 1989). Accordingly,workers skilled in the art can use a combination of synthetic chemistryand enzymatic synthesis to make pharmaceuticals or diagnostic reagents.

Protein fragments of the ligand can also be administered as apharmaceutically acceptable acid- or base- addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamies.

Carbohydrates that are biologically active are those which inhibitbinding of leukocytes to P-selectin. Suitable pharmaceutical vehiclesfor administration to a patient are known to those skilled in the art.For parenteral administration, the carbohydrate will usually bedissolved or suspended in sterile water or saline. For enteraladministration, the carbohydrate will be incorporated into an inertcarrier in tablet, liquid, or capsular form. Suitable carriers may bestarches or sugars and include lubricants, flavorings, binders, andother materials of the same nature. The carbohydrate can also beadministered locally at a wound or inflammatory site by topicalapplication of a solution or cream.

Alternatively, the carbohydrate may be administered in, on or as partof, liposomes or microspheres (or microparticles). Methods for preparingliposomes and microspheres for administration to a patient are known tothose skilled in the art. U.S. Pat. No. 4,789,734 describe methods forencapsulating biological materials in liposomes. Essentially, thematerial is dissolved in an aqueous solution, the appropriatephospholipids and lipids added, along with surfactants if required, andthe material dialyzed or sonicated, as necessary. A good review of knownmethods is by G. Gregoriadis, Chapter 14. “Liposomes”, Drug Carriers inBiology and Medicine pp. 287-341 (Academic Press, 1979). Microspheresformed of polymers or proteins are well known to those skilled in theart, and can be tailored for passage through the gastrointestinal tractdirectly into the bloodstream. Alternatively, the carbohydrate can beincorporated and the microspheres, or composite of microspheres,implanted for slow release over a period of time, ranging from days tomonths. See, for example, U.S. Pat. No. 4,906,474, 4,925,673, and3,625,214.

The carbohydrates should be active when administered parenterally inamounts above about 1 μg/kg of body weight. For treatment of mostinflammatory disorders, the dosage range will be between 0.1 to 30 mg/kgof body weight. A dosage of 70 mg/kg may be required for some of thecarbohydrates characterized in the examples.

The criteria for assessing response to therapeutic modalities employingantibodies or carbohydrate is dictated by the specific condition andwill generally follow standard medical practices. For example, thecriteria for the effective dosage to prevent extension of myocardialinfarction would be determined by one skilled in the art by looking atmarker enzymes of myocardial necrosis in the plasma, by monitoring theelectrocardiogram, vital signs, and clinical response. For treatment ofacute respiratory distress syndrome, one would examine improvements inarterial oxygen, resolution of pulmonary infiltrates, and clinicalimprovement as measured by lessened dyspnea and tachypnea. For treatmentof patients in shock (low blood pressure), the effective dosage would bebased on the clinical response and specific measurements of function ofvital organs such as the liver and kidney following restoration of bloodpressure. Neurologic function would be monitored in patients withstroke. Specific tests are used to monitor the functioning oftransplanted organs; for example, serum creatinine, urine flow, andserum electrolytes in patients undergoing kidney transplantation.

Modifications and variations of the present invention, methods formodulating binding reactions involving P-selectin using carbohydratederived from or forming a portion of the P-selectin ligand, orantibodies to the ligand, will be obvious to those skilled in the artfrom the foregoing detailed description. Such modifications andvariations are intended to come within the scope of the appended claims.

We claim:
 1. A method for inhibiting reperfusion injury comprisingadministering an effective amount of an antibody to a protein componentor to a carbohydrate-protein component of P-selectin glycoproteinligand, the glycoprotein ligand comprising a fucosylated sialylatedglycoprotein containing sialyl Lewis^(x) antigen and the glycoproteinligand having an apparent relative molecular weight of 120,000 asassessed by SDS-PAGE under reducing conditions and wherein the antibodyhas binding specific for P-selectin glycoprotein ligand and wherein theantibody inhibits binding of P-selectin glycoprotein ligand toP-selectin.